Alpha-modified Eagle's Medium Research Articles

Proliferation and Osteoblast Differentiation Mice Dental Pulp Stem Cells between Enzyme Digestion and Outgrowth Method

The isolation method for dental pulp stem cells (DPSCs) is still unclear to obtain a conducive environment for DPSCs to proliferate. Enzymatic digestion and outgrowth method are two commonly used methods for DPSCs isolation but are not well characterized in mice DPSCs. This study aimed to compare these isolation methods and differentiation potential of mice DPSCs into bone cells. Dental pulp was extracted from mice’s incisors and subjected to isolation either by collagenase 1A or culture of pulp tissue in complete alpha-Modified Eagle Medium (αMEM). Both cells isolated were cultured until passage 4 and subjected to in vitro proliferation and differentiation analysis. Both cells exhibited fibroblastliked morphology, but cells isolated by enzyme digestion proliferate faster compare to outgrowth method. After 21 days of osteoblast differentiation, DPSCs isolated from enzyme digestion method showed alkaline phosphatase (ALP) activity slightly different as compared to outgrowth method. In conclusion, there is a significant difference between the cells isolated from enzyme digestion compare to outgrowth method with regard to proliferation and osteoblast differentiation. Thus, it is preferable to isolate by enzyme digestion as it is faster and consistent compared to outgrowth method.

* Efficacy of a Self-Assembling Peptide Hydrogel, SPG-178-Gel, for Bone Regeneration and Three-Dimensional Osteogenic Induction of Dental Pulp Stem Cells.

The aim of this study was to assess the efficacy of a self-assembling peptide hydrogel as a scaffold for bone regeneration. We used a neutral and injectable self-assembling peptide hydrogel, SPG-178-Gel. Bone defects (5 mm in diameter) in rat calvarial bones were filled with a mixture of alpha-modified Eagle's medium and peptide hydrogel. Three weeks after surgery, soft X-ray and microcomputed tomography (micro-CT) images of the gel-treated bones showed new bone formations in the periphery and in central areas of the defects. Next, we evaluated the three-dimensional osteogenic induction of dental pulp stem cells (DPSCs), a type of mesenchymal stem cell, in SPG-178-Gel. We first confirmed that the osteogenic differentiation of DPSCs was significantly promoted by osteogenic induction medium containing recombinant human bone morphogenetic protein-4 (rhBMP-4) in a two-dimensional cell culture. Then, we verified DPSC proliferation and osteogenic differentiation in a three-dimensional cell culture using SPG-178-Gel. The gene expression levels of osteopontin, osteocalcin, and collagen type I were significantly increased when DPSCs were cultured in SPG-178-Gel with the osteogenic induction medium. Micro-CT observations showed the formation of widespread calcium deposition. In conclusion, SPG-178-Gel was adequately effective as a scaffold and can be a suitable tool for bone formation in vivo and in vitro. These findings suggest that the self-assembling peptide hydrogel, SPG-178-Gel, could be a promising tool for bone tissue engineering.

Defined Xenogeneic-Free and Hypoxic Environment Provides Superior Conditions for Long-Term Expansion of Human Adipose-Derived Stem Cells

Development and implementation of therapeutic protocols based on stem cells or tissue-engineered products relies on methods that enable the production of substantial numbers of cells while complying with stringent quality and safety demands. In the current study, we aimed to assess the benefits of maintaining cultures of adipose-derived stem cells (ASCs) in a defined culture system devoid of xenogeneic components (xeno-free) and hypoxia over a 49-day growth period. Our data provide evidence that conditions involving StemPro mesenchymal stem cells serum-free medium (SFM) Xeno-Free and hypoxia (5% oxygen concentration) in the culture atmosphere provide a superior proliferation rate compared to a standard growth environment comprised of alpha-modified Eagle medium (A-MEM) supplemented with fetal calf serum (FCS) and ambient air (20% oxygen concentration) or that of A-MEM supplemented with FCS and hypoxia. Furthermore, a flow cytometric analysis and in vitro differentiation assays confirmed the immunophenotype stability and maintained multipotency of ASCs when expanded under xeno-free conditions and hypoxia. In conclusion, our data demonstrate that growth conditions utilizing a xeno-free and hypoxic environment not only provide an improved environment for the expansion of ASCs, but also set the stage as a culture system with the potential broad spectrum utility for regenerative medicine and tissue engineering applications.

The Use of Human Mesenchymal Stem Cell–Derived Feeder Cells for the Cultivation of Transplantable Epithelial Sheets

To report the efficacy of human bone marrow-derived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets. Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in alpha-modified Eagle's medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithelial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air-liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cytokeratin 3 (K3), K15, p63alpha, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and transplanted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochemistry against K3 and K4. MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63alpha, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits. MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.

Female hormone receptors are differentially expressed in mouse fibrocartilages

Despite the female predilection for joint diseases, and the known effects of female hormones in regulating chondrocyte function, the various female hormone receptor subtypes in joints are not well characterized, and comparisons in receptor profiles between joints and genders are lacking. This investigation characterized and compared the relative levels of estrogen receptors (ER)-alpha and -beta, relaxin receptors LGR7 and LGR8, and progesterone receptor (PR) in the temporomandibular joint (TMJ) disc, knee meniscus (KM) and pubic symphysis fibrocartilages. Fibrocartilaginous cells from 12-week-old mice were maintained in serum-containing alpha-modified Eagle's medium (MEM) until confluence. Total RNA and cell lysates were assayed by RT-PCR, qRT-PCR, immunocytochemistry and Western blots, and joint sections subjected to immunohistochemistry. All hormone receptors assayed were present in the three joints, but showed substantial differences in expression levels between joints. TMJ cells had higher ER-alpha (>2.8-fold), ER-beta (>2.2-fold), LGR7 (>3-fold) and PR (>1.8-fold), and lower LGR8 (0.5-fold) gene expression levels than KM cells. The ratio of ER-alpha:ER-beta and LGR7:LGR8 was 1.8- and 7.5-fold higher, respectively, in TMJ than in KM cells. The profile of hormone receptors in the TMJ disc was similar to those in the pubic symphysis. Immunochemistry confirmed the differential expression patterns of these receptors in the three tissues. The TMJ cells demonstrated sexual dimorphism in the levels of both ER isoforms, but not of LGR7, LGR8 or PR. The findings suggest that these fibrocartilages are putative target tissues for actions of female hormones. The differential expression profiles of the hormone receptors in the three joint fibrocartilages and the sexual dimorphism in ERs in TMJ disc cells are likely to result in varied downstream effects in response to hormones within these fibrocartilaginous tissues.

Nuclear localization of the type 1 parathyroid hormone/parathyroid hormone-related peptide receptor in MC3T3-E1 cells: association with serum-induced cell proliferation

We have recently demonstrated that the receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), PTHR, can be localized to the nucleus of cells within the liver, kidney, uterus, gut, and ovary of the rat. We set out to determine the localization of the PTHR in cultured osteoblast-like cells. MC3T3-E1, ROS 17/2.8, UMR106, and SaOS-2 cells were cultured in alpha-modified eagle medium containing 15% fetal calf serum under standard conditions. Untreated cells were grown on glass coverslips to 75–95% confluence and fixed in 1% paraformaldehyde. For experiments designed to examine cells synchronized by serum starvation, cells were grown on glass coverslips, starved of serum for 46 h, and then fixed at 2-h intervals for a total of 26 h after the addition of serum to the medium. Parallel sets of cells were pulsed with [ 3H]thymidine to track the DNA duplication interval. The PTHR was localized by immunocytochemistry using a primary antibody raised against a portion of the N-terminal extracellular domain of the PTHR. The results presented herein indicate that the PTHR attains a nuclear localization in each cell line examined. In UMR106 cells, PTHR immunoreactivity was restricted to the nucleolus. After cell synchronization, MC3T3-E1 cells double approximately 24 h after the addition of serum. Immunocytochemistry for the PTHR in these cells showed that the receptor staining is initially diffuse for the first 6 h, then becomes more perinuclear in distribution by 12–16 h. Nuclear localization of the receptor is achieved approximately 16–20 h after the addition of serum and remains there throughout the mitotic phase. Intense staining of mitotic and postmitotic cells was observed. No change in cell proliferation kinetics was observed in MC3T3-E1 cells cultured in the presence of 25 nM PTH(1–34). These data suggest an important role for the PTHR in the nucleus of MC3T3-E1 cells at the time of DNA synthesis and mitosis.

Modulation of Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Release in Cultured Male Equine Anterior Pituitary Cells by Gonadal Steroids1

The objective of the present study was to determine whether the testicular steroids, i.e., testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), and estrone sulfate (E1SO4), play a physiological role in regulating LH release in the male horse by direct actions at the anterior pituitary gland. Enzymatically dispersed anterior pituitary cells from stallions (n = 4) or geldings (n = 3) were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse medium. To determine the effects of the steroids on the LH response to GnRH, the cells were incubated for 24 h in fresh media with or without 10(-10) M E2 or 10(-8) M T or DHT followed by a 4-h incubation +/- GnRH (10(-11) to 10(-7) M). Media and cells were analyzed for LH by RIA. In the stallion, GnRH increased LH release (p < 0.001) in a dose-dependent manner (ED50 GnRH = 4.5 x 10(-9) M), and this response was unaltered by T or DHT but greatly enhanced by E2 (p < 0.001). E2 lowered the ED50 for GnRH to 5 x 10(-10) M and increased the maximum LH response to GnRH by 350%. The LH release in response to a constant dose of 1 nM GnRH was unaltered by varying doses of T, DHT, or E1SO4 (10(-11) to 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential Actions of Corticosterone on Luteinizing Hormone and Follicle-Stimulating Hormone Biosynthesis and Release in Cultured Rat Anterior Pituitary Cells: Interactions with Estradiol1

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)

焼成骨の歯周治療への応用 ＩＩ 骨移植材へのコラーゲンコーティングの効果（Ｉｎ ｖｉｔｒｏ）

In vitro studies with osteoblast-like cells, revealed that in addition to preventing the immediate outflow and promoting stabilization of implant materials, these cells have the additional effect of promoting the mending and early calcification by coating the materials with collagen. The materials used for the experiment were minute hydroxyapatite, beta-tricalcium phosphate and bovine sintered bone. To these, atelocollagen which is solble in pepsin extracted from calf corium and crosslinked by using ultraviolet rays was added. We observed the cells very closely after these materials were added to alpha-modified eagle's medium containing 10 mM beta-glycero phosphate, 10% fetal bovine serum and osteoblast-like cells and the cultures incubated at 37 degrees C and 5% CO2. After 14 and 21 days, cells were fixed and stained with Alizarin Red S and Von Kossa stains to measure calcification. As a result of the collagen coating, positive areas appeared and, compared to the control improvement of the early mending was apparent. The results suggest that by coating the materials with collagen, the osteoblast-like cells show better mending and early calcification.